Download Genome Rearrangements Caused by Depletion of Essential DNA

INVESTIGATION
Genome Rearrangements Caused by Depletion
of Essential DNA Replication Proteins
in Saccharomyces cerevisiae
Edith Cheng,*,† Jessica A. Vaisica,*,† Jiongwen Ou,*,† Anastasia Baryshnikova,†,‡ Yong Lu,§
Frederick P. Roth,†,§,** and Grant W. Brown*,†,1
*Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada, †Donnelly Centre for Cellular and
Biomolecular Research, Toronto, Ontario M5S 3E1, Canada, ‡Banting and Best Department of Medical Research and Department
of Molecular Genetics, University of Toronto, Toronto, Ontario M5G 1L6, Canada, §Department of Biological Chemistry and
Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, and **Samuel Lunenfeld Research Institute, Mt.
Sinai Hospital, Toronto, Ontario M5G 1X5, Canada
ABSTRACT Genetic screens of the collection of 4500 deletion mutants in Saccharomyces cerevisiae have identified the cohort of
nonessential genes that promote maintenance of genome integrity. Here we probe the role of essential genes needed for genome
stability. To this end, we screened 217 tetracycline-regulated promoter alleles of essential genes and identified 47 genes whose
depletion results in spontaneous DNA damage. We further showed that 92 of these 217 essential genes have a role in suppressing
chromosome rearrangements. We identified a core set of 15 genes involved in DNA replication that are critical in preventing both
spontaneous DNA damage and genome rearrangements. Mapping, classification, and analysis of rearrangement breakpoints indicated
that yeast fragile sites, Ty retrotransposons, tRNA genes, early origins of replication, and replication termination sites are common
features at breakpoints when essential replication genes that suppress chromosome rearrangements are downregulated. We propose
mechanisms by which depletion of essential replication proteins can lead to double-stranded DNA breaks near these features, which
are subsequently repaired by homologous recombination at repeated elements.
A
CCURATE transmission of the genome is essential for
normal cell growth and survival. As such, cells have
developed elaborate mechanisms to prevent errors in replication and to respond to spontaneous DNA damage that can
lead to genomic instability (Kolodner et al. 2002; Branzei
and Foiani 2007, 2009, 2010; Harper and Elledge 2007;
Cimprich and Cortez 2008). The failure to repair the genome
in an error-free manner can result in chromosome abnormalities that underlie many human diseases, including cancers
(Kolodner et al. 2002; McKinnon and Caldecott 2007; Aguilera
and Gomez-Gonzalez 2008). Therefore, defining the genes
that contribute to genome maintenance will be useful in
understanding disease development and in designing new
strategies for therapeutics. However, to date, a comprehensive
Copyright © 2012 by the Genetics Society of America
doi: 10.1534/genetics.112.141051
Manuscript received April 9, 2012; accepted for publication May 28, 2012
Supporting information is available online at http://www.genetics.org/content/
suppl/2012/06/05/genetics.112.141051.DC1.
1
Corresponding author: Donnelly Centre for Cellular and Biomolecular Research, 160
College St., Toronto, ON M5S 3E1, Canada. E-mail: [email protected]
curation of genes that function to suppress genome instability is incomplete.
Yeast is an ideal model for genomic studies due to the
conservation of gene functions and biological pathways between yeast and humans. Phenotypic screens conducted with
the Saccharomyces cerevisiae nonessential gene deletion collection (Giaever et al. 2002) have aided in the annotation and
functional characterization of nonessential genes involved in
the suppression of spontaneous DNA damage (Huang et al.
2003; Huang and Kolodner 2005; Shor et al. 2005; Alvaro
et al. 2007) and in the suppression of spontaneous chromosome
rearrangements (Smith et al. 2004; Yuen et al. 2007; Andersen
et al. 2008). However, since the deletion of essential genes
causes lethality, similar genome-wide screening approaches to
identify the complete set of genes that suppress spontaneous
DNA damage and chromosome rearrangements require collections of conditional alleles of essential genes.
Systematic collections of conditional alleles have been
generated in several ways, including the replacement of native
promoters with a tetracycline-regulated promoter (Mnaimneh
Genetics, Vol. 192, 147–160 September 2012
147
et al. 2004; Yu et al. 2006), destabilization of target gene
mRNAs through the insertion of a selectable marker in the
39-UTR of essential genes (Schuldiner et al. 2005), systematic
addition of a heat-inducible degron to the amino terminus of
the protein product (Labib et al. 2000), systematic generation
of novel temperature-sensitive alleles (Ben-Aroya et al.
2008), and systematic integration of existing temperaturesensitive alleles (Li et al. 2011). Despite the availability of
several essential gene collections, no one collection is complete, suggesting that complementary approaches using
a number of screening strategies and multiple types of conditional alleles will be necessary to identify all of the essential genes that function to suppress genomic instability.
Here we describe a series of screens to identify essential
genes that function to suppress genome instability, using the
collection of tetracycline-regulated promoter replacement
alleles (Tet alleles) of essential genes (Mnaimneh et al.
2004). We screened 217 Tet alleles of essential genes whose
depletion caused accumulation in S or G2 phases of the cell
cycle (Yu et al. 2006) and identified 47 with elevated levels
of spontaneous DNA damage. A second screen performed
with the same Tet alleles identified 92 essential genes that
suppress the formation of chromosome rearrangements, whole
chromosome deletions, and gene conversions. We quantified
the levels of each type of mutation in 15 strains that exhibited
both elevated levels of spontaneous DNA damage and chromosome rearrangements following the depletion of an essential gene. Mapping of rearrangement breakpoints in seven
representative mutants from this set revealed several unique
rearrangement structures. Sequence features, including Ty retrotransposons and DNA replication origins and termination
zones, correlated with the rearrangements identified. We propose a central role for DNA replication proteins in suppressing
the formation of chromosome breaks that promote chromosome rearrangements.
Materials and Methods
Yeast strains and media
Tet allele strains were constructed as described previously
(Mnaimneh et al. 2004). The genotype of the wild-type
Tet allele strain, R1158, is MATa URA3::CMV-tTA his3D1
leu2D0 met15D0. Using standard genetic methods, 217 MATa
Tet allele strains were engineered to contain YFP-Ddc2
marked with a nourseothricin (Nat) resistance gene. Genotypes for strains used in this study are listed in Table S6.
The essential genes that were studied are listed in Table S1
and Table S2. Standard yeast media and growth conditions
were used unless otherwise specified (Sherman 1991).
Fluorescence microscopy
Tet allele strains were grown in YPD liquid media at 30.
Samples were divided into two cultures and grown in parallel in the presence and absence of 10 mg/ml doxycycline
for 4 additional hours at 23. Intracellular localization of
Ddc2-YFP was determined by fluorescence microscopy as
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E. Cheng et al.
previously described for Rad52-YFP (Lisby et al. 2004; Lisby
and Rothstein 2004; Chang et al. 2005). Ddc2 foci were
quantified in at least 100 cells for each strain. Ddc2 foci in
wild-type cells were analyzed four times and used to calculate a standard deviation. Tet allele strains that had Ddc2
foci levels that were at least three standard deviations greater
than wild type were scored as positive.
Illegitimate mating assays
Tet allele strains and the R1158 wild-type strain were grown
in parallel for 24 hr on YPD solid media either containing or
lacking 10 mg/ml of doxycycline. A standard mating assay
was performed with tester strains MCY13 (MATa , legitimate mating) and MCY14 (MATa, illegitimate mating) on
the same media conditions that the strains were grown.
Diploids were isolated by replica plating on minimal media.
In the quantitative form of this mating assay, Tet allele
strains and R1158 wild-type strain were grown in parallel
for 24 hr in YPD liquid media containing or lacking 10 mg/ml
doxycycline. Strains were mixed with fivefold excess of
MCY13, MCY14, or 1225a (MATa his4 thr4) tester strains
and plated on YPD solid media. After 5 hr, cells were collected, resuspended in water, and plated on diploid selection
media. Independent illegitimate diploids were isolated after
the mating of the Tet allele strains with the 1225a tester
strain. For each mating experiment, 100 diploids were isolated and tested for their ability to grow in the presence or
absence of histidine or threonine. This assay was repeated two
times. Viability of each strain following growth in doxycycline
was confirmed by plating on YPD. Only MCM7 (10%), NUF2
(30%), and UBC9 (50%) had ,100% viability following
growth in doxycycline.
Array comparative genome hybridization
Genomic DNA was extracted (Qiagen) from independent
illegitimate diploids and wild-type diploids isolated from the
mating assay. CGH on a microarray was performed as
previously described (Dion and Brown 2009) using S. cerevisiae
whole genome tiling microarrays (Affymetrix). Signal intensities of the experimental and wild-type control samples were normalized and compared using tiling analysis
software (Affymetrix). Genomic patterns were mapped and
analyzed using the integrated genome browser software
(Affymetrix).
CHEF gel electrophoresis and Southern blot analysis
Contour-clamped homogenous electric field (CHEF) gels
were used to examine intact chromosomes of illegitimate
diploids isolated from the mating assay. CHEF gel analysis
was performed as described previously (Desany et al.
1998). A 1.2% agarose gel was run at 8 V/cm using pulse
times of 120 sec for 30 hr at 14 in 0.5· TBE buffer. PCRpurified fragments were radio labeled by random priming
(Stratagene) and used as hybridization probes for Southern
blot analysis. PCR primers designed for probe construction
are listed in Table S7.
Figure 1 Depletion of yeast essential genes results in elevated levels of spontaneous Ddc2 foci formation. (A) A
total of 217 Tet alleles that express Ddc2-YFP and display
a G2/M or S phase cell cycle arrest phenotype were grown
in the presence of doxycycline (10 mg/ml) for 4 hr to inhibit
the transcription of each essential gene. Representative
DIC and YFP images are shown for the wild-type, DPB11
and NSE1 strains. Ddc2-YFP foci are indicated with white
arrows. (B) The percentage of cells with Ddc2-YFP foci is
plotted for 47 Tet alleles that showed an increase in Ddc2
foci of at least three standard deviations above the average observed in wild type. Bars are shaded according to
the GO process annotation of each gene of interest.
Restriction digestion and sequencing analysis
of FS1 and FS2
Genomic DNA was isolated (Qiagen) from wild-type strains
R1158 and BY4741 and digested with EcoRI and XbaI (New
England Biolabs) using the suggested conditions. Digested
fragments were separated on a 1% agarose gel and hybridized with FEN2 and FS2-2 probes for Southern blot analysis (Table S7). 59 and 39 ends of fragile site 1 (FS1) and
FS2 were PCR amplified and sequenced. PCR primers used for
both amplification and sequencing are listed in Table S8.
Enrichment analyses
S. cerevisiae chromosomes were broken into 5-kb bins. For
each bin, the presence or absence of breakpoints and genomic features was tabulated. Various genomic features (Di
Rienzi et al. 2009) and replication termination sites (Fachinetti
et al. 2010) from previous datasets were used for analysis. For
each feature, the total number of bins with both the feature
and a breakpoint was determined. To test for enrichment of
breakpoints and each feature, a hypergeometric distribution was assumed. P-values ,0.05 were considered as evidence of a correlation and P-values ,0.05 after a false
discovery rate (FDR) correction were considered strongly
significant.
Results
Depletion of essential gene products causes
spontaneous DNA damage
We used a collection of tetracycline-regulated promoter
alleles (Tet alleles) (Mnaimneh et al. 2004; Yu et al. 2006)
of essential genes to systematically identify genes that suppress spontaneous DNA damage. Since elevated levels of
spontaneous DNA damage should elicit a checkpoint response and cause cell cycle delay, we screened the 217
strains that accumulated in S phase or G2 phase of the cell
cycle following gene-product depletion by promoter shut
off (Yu et al. 2006). Spontaneous DNA damage was measured by the relocalization of the DNA damage checkpoint
protein Ddc2 from a diffuse nuclear pattern to discrete subnuclear foci (Figure 1A) (Melo et al. 2001; Lisby et al. 2004).
Following growth of these strains in doxycycline to repress
essential gene expression, the fraction of cells with Ddc2 foci
was quantified (Supporting Information, Table S1). We
Essential Genome Stability Genes
149
Figure 2 Depletion of yeast essential genes results in elevated
levels of illegitimate mating. (A)
MATa Tet alleles were grown
on YPD or YPD containing doxycycline (10 mg/ml) for 24 hr and
a standard mating test was performed using MATa and MATa
tester strains. Representative
images of strains with elevated
levels of illegitimate diploid formation following growth in
doxycycline are shown. (B) The
resulting number of illegitimate
diploid colonies that grew without doxycycline treatment was
subtracted from the number that
grew with doxycycline treatment
and was used to subcategorize
the strains into four groups. For
each group, the distribution of
percentage of budded cells with
Ddc2-YFP foci was plotted. Bold
lines represent the median values, boxes represent the upper
and lower quartiles, whiskers
represent 1.5 times the interquartile range, and outliers are
indicated by circles. (C) Comparison of Tet alleles with elevated
levels of Ddc2 foci and .10 illegitimate mating diploid colonies.
determined that the individual depletion of 47 essential
gene products caused an increase of Ddc2 foci relative to
wild-type levels, using a cutoff of three standard deviations from the wild-type mean (Figure 1B). The gene ontology (GO) processes of the essential genes that were
identified are varied (Table S2), but on average the highest
levels of Ddc2 foci were observed following the depletion of
gene products involved in DNA replication, response to DNA
damage stimuli, and cell cycle progression, indicating the importance of these essential processes in the maintenance of
genome integrity (Figure 1B). In addition to the identification
of essential genes with defined roles in genome maintenance,
20 essential genes with previously unrecognized contributions
to the suppression of spontaneous DNA damage were also
identified (Figure 1B, gray bars).
Depletion of essential gene products causes
chromosome loss and rearrangement
Increased levels of Ddc2 foci could reflect increased spontaneous DNA damage, defective repair of spontaneous DNA
damage, or a combination of both. An increase in spontaneous
DNA damage may not impact genome integrity if the damage
is repaired accurately. To directly identify essential genes that
suppress chromosome rearrangements and chromosome loss,
we used an illegitimate mating assay (Strathern et al. 1981;
Lemoine et al. 2005, 2008) that measures loss of genetic
information from chromosome III to screen the same 217
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E. Cheng et al.
Tet allele strains. Mutation or deletion of the MATa locus
on chromosome III in haploid cells results in a reversion to
the default MATa mating type, termed a-like fakers, allowing these MATa cells to mate illegitimately with strains of
the MATa mating type (Strathern et al. 1981). We determined
the levels of a-like faker formation using a patch mating assay
(Figure 2A). We found that the depletion of 92 essential genes
caused elevated illegitimate mating frequencies both relative
to the minus doxycycline control and relative to the wild-type
control, indicating loss of genetic information at the MAT locus
in these strains (Table S3). Thirty strains did not form colonies
in the presence of doxycycline and 9 strains could not be
constructed with the MATa mating type and therefore could
not be evaluated. Strains were subcategorized into groups
with high (.10 colonies; 57 strains), moderate (1–10 colonies; 35 strains), or wild-type (0 colonies; 86 strains) levels of
illegitimate mating and the distributions of Ddc2 foci formation for each category were compared (Figure 2B). Both the
high and medium categories had greater Ddc2 foci formation when compared to the wild-type category (P-value of
0.022 for high vs. wild type and P-value of 0.028 for medium vs. wild type; one-sided Mann–Whitney test), indicating a relationship between the extents of Ddc2 focus
formation and the illegitimate mating phenotype. Additionally, strains with spontaneous Ddc2 foci formation
above our cutoff were more likely to have increased illegitimate mating (P-value of 0.00073; hypergeometric test),
Table 1 Frequencies of illegitimate mating in tetracyclineregulatable promoter conditional alleles grown in the presence
and absence of doxycycline
Frequencies of illegitimate mating (1024)
2doxycycline
Tet allele
Wild type
CDC45
CSE1
DNA2
DPB11
MCM4
MCM5
MCM7
NSE1
NUF2
POL2
POL30
RFC2
RFC5
SPT16
UBC9
0.83
11
0.5
4.3
0.94
11
3.4
4.1
17
2.5
2.2
5.4
2.2
1.5
2.4
2.4
(0.20)
(6.0)
(0.04)
(2.7)
(0.1)
(9.8)
(0.8)
(2.9)
(11)
(1.1)
(0.9)
(4.9)
(0.03)
(0.76)
(1.7)
(0.4)
+doxycycline
[1]
[14]
[0.6]
[5.2]
[1.1]
[13]
[4.1]
[4.9]
[21]
[3.0]
[2.6]
[6.5]
[2.6]
[1.8]
[2.9]
[2.9]
0.8
17
1.4
4.7
9.2
36
12
34
39
18
26
8.3
5.9
3.0
11
3.9
(0.26)
(1.6)
(0.40)
(2.5)
(3.4)
(27)
(3.0)
(24)
(7.3)
(8.9)
(21)
(2.7)
(0.14)
(0.64)
(11)
(2.2)
[1]
[22]
[1.8]
[5.9]
[12]
[45]
[15]
[62]
[48]
[22]
[33]
[10]
[7.4]
[3.8]
[14]
[4.8]
Tet allele strains and wild-type strain were grown in parallel for 24 hr on YPD liquid
media containing or lacking 10 mg/ml doxycycline. Strains were mixed with fivefold
excess of a MATa tester strain and plated on YPD solid media. After 5 hr, cells were
resuspended in water and plated on illegitimate diploid selection media. This assay
was repeated two times. Numbers in parentheses represent standard deviations.
Numbers in brackets represent the frequency normalized to wild type.
although the overlap between the two screens, at 29 genes,
was not absolute.
We focused on the strains with the most robust chromosome instability phenotype, the 15 strains with both elevated
Ddc2 foci and high levels of illegitimate mating (Figure 2C).
These strains were subjected to a quantitative illegitimate
mating assay (Table 1). In the presence of doxycycline, all
of these strains exhibited significantly elevated levels of illegitimate mating relative to the wild-type strain. Increases in
illegitimate mating ranged from ,2-fold wild type (CSE1) to
62-fold wild type (MCM7). Previous studies of GAL promoterregulated conditional alleles of DNA polymerases a and d
found increases of 200-fold and 50-fold, respectively
(Lemoine et al. 2005, 2008). Although DNA polymerases a
and d were not assayed in our screens, we identified a role for
DNA polymerase e (POL2 and DPB11) in suppressing illegitimate mating. Additionally, we found that disrupting a wide
range of replication functions (CDC45, MCM4, MCM5, MCM7,
DPB11, POL2, POL30, RFC2, and RFC5) caused increased illegitimate mating. DNA2, which functions in Okazaki fragment
processing (Budd et al. 2000; Lee et al. 2000) and in DNA
repair (Zhu et al. 2008) resulted in increased illegitimate mating, as did repression of the DNA repair genes NSE1 (Santa
Maria et al. 2007; Pebernard et al. 2008) and UBC9 (Branzei
et al. 2006). Genes with functions outside of DNA replication
and repair were also identified. CSE1 is responsible for
nuclear shuttling of the nuclear transporter importin a (Hood
and Silver 1998; Kunzler and Hurt 1998; Solsbacher et al.
1998), and roles for CSE1 in DNA replication (Yu et al.
2006) and in chromosome segregation (Xiao et al. 1993),
likely reflecting effects on importin a cargos, have been described. NUF2 is a kinetochore component and functions in
chromosome segregation (Osborne et al. 1994). Depletion of
Figure 3 Classification of rearrangement events that lead to illegitimate mating. (A) Schematic
diagram of the three expected
classes of genetic events resulting in illegitimate mating. Using
diagnostic selection media, mutations in the MAT locus, whole
chromosome III loss, and loss of
the right arm of chromosome III
can be distinguished as class 1, 2,
and 3 genetic events, respectively
(Lemoine et al. 2005, 2008). (B)
We classified 200 illegitimate
diploids for each strain. The frequencies of the three classes of
rearrangements are plotted for
the 15 strains with the most elevated levels of illegitimate mating.
(C) Ratios of the three classes of
rearrangements are plotted for
the indicated strains.
Essential Genome Stability Genes
151
Figure 4 Comparative genome hybridization
microarray analysis of class 3 illegitimate diploids.
Genomic DNA was isolated from class 3 (chromosome III arm loss) illegitimate diploids and hybridized to a Saccharomyces cerevisiae whole genome
tiling microarray to identify copy number variations.
In each histogram, the y-axis represents log2 ratios
of probe signal intensities, comparing the indicated
strain to a legitimate MATa/a diploid, and the xaxis represents chromosome coordinates. Black
arrows indicate breakpoint locations on each chromosome, black circles represent the locations of
centromeres, and the chromosome number is indicated to the right of each histogram. A representative histogram for each of the major types of
rearrangements observed is shown. (A) Class 3-1
diploid, in which no copy number variation of chromosome III was evident. (B) Class 3-2 diploids have
a loss of sequence (red) from the right arm of chromosome III and duplication of sequences (blue)
from chromosome XV. (C) Class 3-3 diploids have
an amplification of the left arm sequence and a deletion of the right arm sequence of chromosome III.
(D) Class 3-4 diploids have a loss of sequence from
the right arm of chromosome III without copy number variation on nonhomologous chromosomes. (E)
Class 3-5 diploids exhibit a loss of sequence from
the right arm of chromosome III and loss of sequence from the right arm of chromosome V.
DNA replication (CDC45, MCM4, MCM7, and POL2) and segregation (NUF2) gene products had the most striking effect
(.20-fold difference).
Chromosome III rearrangements in essential genome
stability mutants
Three common classes of information loss on chromosome
III in the illegitimate mating assay can be distinguished by
performing the assay with a strain with nutritional markers
flanking the MAT locus on chromosome III (Lemoine et al.
2005) (Figure 3A). We used this modified assay to classify
chromosome instability events in the 15 strains with both
increased spontaneous DNA damage and high levels of illegitimate mating. Class 1 mating events result from a gene
conversion or mutation at the MATa locus. Class 2 events
result from the loss of chromosome III. Class 3 events result
from chromosome rearrangements that lead to the loss of
the MATa locus and distal regions of the right arm of chromosome III (Figure 3A). For each strain we classified the
chromosome rearrangements in 200 illegitimate diploids
and measured the frequencies and ratios of the three classes
(Figure 3, B and C).
Increases in class 2 (whole chromosome loss) and 3
(chromosome arm loss) rearrangements were evident for all
15 genes tested (Figure 3B). Depletion of CSE1, DPB11, MCM4,
MCM5, POL30, SPT16, and UBC9 resulted in ratios of the three
classes of chromosome instability that were not significantly different than that observed in the wild-type strain
(P-value .0.01 by the Fisher exact test) (Figure 3C). This
could indicate that depletion of these gene products exacer-
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E. Cheng et al.
bates a condition already present in wild-type cells. By contrast, repression of CDC45, DNA2, MCM7, RFC2, RFC5, and
POL2 resulted in a significant difference in ratios of the three
classes relative to wild type (P-value ,0.01 by the Fisher exact
test) with a preferential increase in class 3 (chromosome arm
loss) events. Depletion of NUF2, a kinetochore-associated protein, resulted in a dramatic increase in class 2 (whole chromosome loss) events, consistent with the function of this gene
in chromosome segregation (Osborne et al. 1994; Wigge and
Kilmartin 2001). Finally, we observed that the depletion of
NSE1, a subunit of the structural maintenance of chromosome
(Smc5/6) complex (Fujioka et al. 2002), resulted in the loss of
class 1 (gene conversion or point mutation) events and in
similar levels of class 2 (whole chromosome loss) and class
3 (chromosome arm loss) events. Our data suggest that NSE1
contributes to both the DNA repair (class 3) and chromosome
segregation (class 2) functions of the SMC5/6 complex (Santa
Maria et al. 2007; Behlke-Steinert et al. 2009; Irmisch et al.
2009; Outwin et al. 2009). We conclude that depletion of
different essential gene functions can cause different patterns
of genomic instability.
Essential gene product depletion causes genome
rearrangements with boundaries at Ty retrotransposons
To obtain a comprehensive assessment of the chromosome
rearrangement breakpoint locations in the illegitimate diploids
that were isolated following essential gene depletion in our
classification experiment, we used comparative genome hybridization on tiling microarrays to map rearrangement breakpoints (Dion and Brown 2009). Genomic DNA was isolated
from six illegitimate diploid colonies that exhibited a chromosome III arm loss (class 3) phenotype following the depletion of each of seven essential genes (CDC45, DPB11, MCM7,
NSE1, RFC2, SPT16, and UBC9), chosen to represent the functional diversity present in the quantitative illegitimate mating
assay. Genomic DNA was hybridized to a S. cerevisiae whole
genome tiling microarray and copy number variation was determined by comparison to genomic DNA isolated from
a wild-type a/a diploid. Representative copy number profiles are depicted in Figure 4 and the boundaries of each
rearrangement are shown in Table 2. Figure 5, A and B
summarizes the breakpoints observed on chromosome III
and the resulting subclasses of rearrangements that were
observed.
Four of the 42 class 3 illegitimate diploids that we tested
exhibited poor hybridization profiles and were not analyzed
further. The remaining samples were divided into five different
subclasses on the basis of the rearrangement profiles of
chromosome III. The most frequent subclass, class 3-1,
comprised microarray profiles that lacked deletions or duplications (Figure 4A). This type of profile was observed in 15 of
38 (39%) illegitimate diploids and was present following the
depletion of all essential genes tested, with the exception of
RFC2 and SPT16. As previously suggested, this genome profile
likely represents successful repair of chromosome III by breakinduced replication (BIR) using the 1225a strain chromosome
III as a template (Lemoine et al. 2005, 2008), resulting in full
restoration of chromosome III sequences (Figure 5B). Southern blot analysis following separation of chromosomes on
a contour-clamped homogeneous electric field (CHEF) gel,
using a probe specific for the HIS4 locus on the left arm of
chromosome III, revealed a single DNA band corresponding to
the size of chromosome III, confirming that class 3C diploids
have two intact copies of chromosome III (Figure 6A, lane 4).
Table 2 Classification of class 3 illegitimate diploid chromosome rearrangements
Classa
Strain
Essential
gene
depleted
Type of
chromosome
rearrangement
Chromosomes
involved
Size of altered
chromosome (kb)
3A(i)b
MCM7-2-57-1
MCM7
Translocation
III/VII
690
3A(i)
3A(i)
3A(i)b
3A(ii)b
RFC2-1-1-1
RFC2-1-1-2
SPT16-1-3-2
CDC45-4-4-1
RFC2
RFC2
SPT16
CDC45
Translocation
Translocation
Translocation
Ectopic BIR
III/VII
III/VII
III/XV
III/III
710
710
640
120
3A(ii)
CDC45-4-52-1
CDC45
Ectopic BIR
III/III
140
3A(ii)b
MCM7-6-1-1
MCM7
Ectopic BIR
III/III
140
3A(ii)
RFC2-2-56-1
RFC2
Ectopic BIR
III/III
140
3A(ii)
RFC2-2-6-1
RFC2
Ectopic BIR
III/III
120
3A(ii)
RFC2-2-6-2
RFC2
Ectopic BIR
III/III
120
3A(ii)
SPT16-1-55-1
SPT16
Ectopic BIR
III/III
120
3A(ii)b
SPT16-2-1-1
SPT16
Ectopic BIR and
interstitial deletion
III/III and V
170 (chrIII)
520 (chrV)
3Bb
3B
3B
3B
3Db
3Fb
DPB11-1-1-2
MCM7-4-1-2
SPT16-2-51-1
SPT16-2-51-2
RFC2-1-52-1
DPB11-2-56-1
DPB11
MCM7
SPT16
SPT16
RFC2
DPB11
Arm deletion
Arm deletion
Arm deletion
Arm deletion
Hawthorne deletion
Chromosome fusion
III
III
III
III
III
III/XVI
150
170
120
120
220
970
3Fb
3F
3Fb
NSE1-4-5-1
NSE1-6-51-1
UBC9-1-51-1
NSE1
NSE1
UBC9
Chromosome fusion
Chromosome fusion
Chromosome fusion
III/V
III/V
III/XVI
530
580
930
3F
UBC9-1-51-2
UBC9
Chromosome fusion
III/XVI
930
3F
UBC9-1-51-3
UBC9
Chromosome fusion
III/XVI
930
a
b
Boundaries of
chromosome
rearrangements
FS2;
YGRCTy1-2, YGRCTy2-1
FS1; YGRWTy1-1
FS1; YGRWTy1-1
FS1; YORWTy1-2
YCLWTy2-1;
YCRCd6
YCLWTy2-1;
YCRCd7
YCLWTy2-1;
YCRCd7
YCLWTy2-1;
YCRCd7
YCLWTy2-1;
YCRCd6
YCLWTy2-1;
YCRCd6
YCLWTy2-1;
YCRCd6
YCLWTy2-1;
FS2 (chrIII) and YERCTy1-1;
YERCTy1-2 (chrV)
FS1
FS2
YCRCd6
YCRCd6
MATa, HMR
YCLWTy2-1;
YPLWTy1-1
YCLWTy2-1; YERCTy1-1
YCLWTy2-1; YERCTy1-2
YCLWTy2-1; YPRWTy1-3,
YPRCTy1-4
YCLWTy2-1; YPRWTy1-3,
YPRCTy1-4
YCLWTy2-1; YPRWTy1-3,
YPRCTy1-4
All strains were examined by comparative genome hybridization on a microarray.
Genome rearrangements predicted from microarray analysis were confirmed by Southern blot analysis.
Essential Genome Stability Genes
153
Figure 5 Mechanisms of repair in replication deficient mutants. (A) Schematic of boundaries of
rearrangement that occur on chromosome III of
replication mutants are shown. (B) Following a
double stranded break and resection to Ty retrotransposons (arrows) or d long terminal repeats (triangles), chromosome fragments can be repaired in
illegitimate diploids through several mechanisms.
Class 3-1: Chromosome III is repaired by BIR using
the homologous chromosome of the tester strain.
Class 3-2: Ectopic break-induced replication (BIR)
mediated by strand invasion at a Ty retrotransposon on a nonhomologous chromosome XV of the
tester strain (gray) yields nonreciprocal translocations. Class 3-3: Ectopic BIR involving strand invasion at a different locus of chromosome III results in
shortened fragments of chromosome III with two
left arms. Class 3-4: Chromosome III fragments are
directly repaired by telomere acquisition. Class 3-5:
Chromosome fusions can be created through single stranded annealing (SSA) of chromosome III
and chromosome V fragments with boundaries at
Ty retrotransposons or through a BIR and halfcrossover event.
Class 3-2 diploids had both a deletion of sequences from
the right arm of chromosome III and a duplication of sequences
from a non-homologous chromosome, suggesting the presence
of a nonreciprocal chromosome translocation (Figures 4B and
5B). Illegitimate diploids isolated following the depletion of
MCM7, RFC2, and SPT16 displayed this type of rearrangement.
As previously described following depletion of DNA polymerase a or d (Lemoine et al. 2005, 2008), this class had
breakpoints either at FS1 (Fragile Site 1) or FS2 of chromosome III and at Ty1 retrotransposons of a nonhomologous
chromosome (chromosome VII or XV, in our case). Previous
sequencing analyses and restriction mapping of FS1 and FS2
regions in several strain backgrounds, including the S288C
strain background that we used in our study, indicated the
presence of a direct pair of tandem Ty1 retrotransposons
and an inverted pair of Ty1 retrotransposons, respectively, that
are unannotated in the Saccharomyces Genome Database
(SGD) (Umezu et al. 2002; Lemoine et al. 2005, 2008; Hoang
et al. 2010). We confirmed that this same arrangement of Ty1
retrotransposons at FS1 and FS2 is present in the wild-type
Tet allele strain using PCR analyses (Figure S1A). We further
verified the arrangement of Ty1 retrotransposons at FS2 using
Southern blot analyses (Figure S1B). In keeping with the
mechanism proposed by Lemoine et al. (2005, 2008), we predict that class 3-2 represents ectopic BIR with strand invasion
at a Ty1 retrotransposon element of a nonhomologous chromosome (Figure 5B). Southern blot analysis of a representative SPT16 illegitimate diploid revealed one band consistent
with the size of chromosome III as well as a second band
consistent with the predicted size of a nonreciprocal translocation between chromosome III and chromosome XV (Figure
6B, lane 2). Hybridization of the same blot with a chromosome
XV probe confirmed the presence of a chromosome containing
sequences from both chromosome III and chromosome XV
(Figure 6B, lane 4).
154
E. Cheng et al.
Class 3-3 illegitimate diploids had both an amplification
of sequences proximal to the Ty2 retrotransposon, YCLWTy21, and a deletion of right arm sequences distal to FS2 or d
elements YCRCd6 or YCRCd7 of chromosome III (Figures 4C
and 5B). Although class 3-2 was the most common class of
rearrangement following depletion of DNA polymerase a or d
(Lemoine et al. 2005, 2008), class 3-3 was more common in
our study. Sequencing of chromosome III in the S288C strain
background used in our study indicates a Ty1 retrotransposon
insertion directly upstream of YCLWTy2-1 in the forward orientation that is unannotated in the SGD, resulting in a configuration similar to FS1 (Hoang et al. 2010). This was the
second most abundant profile and was observed in eight samples, following the depletion of CDC45, MCM7, RFC2, or
SPT16. One model for this rearrangement (Figure 5B) is that
it results from a BIR event with inaccurate strand invasion at
the Ty retrotransposons on the left arm of chromosome III,
which have homology to the Ty retrotransposons at the breakpoints on the right arm of chromosome III. The high frequency
of this type of rearrangement could reflect the abundance and
orientation of long terminal repeats (LTRs) and Ty retrotransposons on chromosome III or the spatial proximity of the
relevant Ty retrotransposons and LTRs within the nucleus
(Duan et al. 2010). Southern blot analysis using a probe
within the amplified region of chromosome III resulted in
the detection of two distinct chromosome sizes (Figure 6A,
lane 2). One corresponds to the expected size of an intact
chromosome III contributed by the tester strain and the other
(of lower molecular weight) corresponds to the size of the
inaccurately repaired chromosome predicted by comparative
genome hybridization (CGH) data. Additionally, the intensity
of the rearranged chromosome band was approximately twofold higher than that of the intact chromosome III band. Band
sizes and intensities are consistent with the position of the
probe in the amplified region of the left arm and therefore
Figure 6 Southern blot confirmation of chromosome rearrangements
predicted in microarray analysis. (A) Intact chromosomal DNA was isolated from class 3 (chromosome arm loss) illegitimate diploids. Genomic
DNA was separated on a contour-clamped homogenous electric field
(CHEF) gel and chromosome III was detected through hybridization with
a radio-labeled probe specific to the left arm of chromosome III. Smaller
chromosome III fragments were also detected in samples with chromosome rearrangements. Representative Southern blots for a wild-type diploid, classes 3-3, 3-4, and 3-1 diploids of the indicated strains are shown,
respectively. (B) Representative Southern blots of a wild-type and a class
3-2 illegitimate diploid. Chromosomes on the left and right were detected
with probes for chromosomes III and XV, respectively. In addition to
chromosome III and XV, a nonreciprocal translocation (nrt) was visualized.
(C) Representative Southern blot of a wild-type and a class 3-5 illegitimate
diploid. Chromosomes on the left and right were detected with probes
for chromosome III and V, respectively. In addition to chromosomes III and
V, a chromosome fusion (fus) event was detected.
support the indicated structure of this class of rearrangement
(Figure 5B).
Class 3-4 illegitimate diploids have only a deletion of the
right arm of chromosome III (Figures 4D and 5B). We observed four of these events after the depletion of genes involved in DNA replication (MCM7, DPB11, and SPT16). Two
of these events had breakpoints at FS1 and FS2 (MCM7 and
DPB11), the same breakpoints observed following depletion
of DNA polymerases a and d (Lemoine et al. 2005, 2008).
The other two, from SPT16 illegitimate diploids, had breakpoints at YCRCd6. As suggested previously, this class might
represent chromosome fragments that persist through direct
telomere capping (Figure 5B) or by acquisition of telomeric
sequences by BIR utilizing a telomere-proximal Ty element
(Lemoine et al. 2005, 2008). These rearrangement profiles
were confirmed by Southern blot analysis, where two bands
of equal intensity were visualized, one corresponding to intact chromosome III and the other to the predicted chromosome III fragment (Figure 6A, lane 3).
The class 3-5 rearrangement pattern includes a deletion
of all but the left arm of chromosome III in addition to an
arm deletion on a nonhomologous chromosome (chromosome
XVI or V) (Figures 4E and 5B). By contrast to the four subclasses of rearrangements described above, class 3-5 profiles
were not observed following depletion of DNA polymerases a
and d (Lemoine et al. 2005, 2008). However, this profile has
been documented upon the deletion of a nonessential gene
required for strand invasion in homologous recombination,
RAD52 (Casper et al. 2009). In our studies, class 3-5 profiles
were observed following the depletion of NSE1 and UBC9,
which also function in DNA repair (Branzei et al. 2006; Santa
Maria et al. 2007; Irmisch et al. 2009). We found one additional class 3-5 rearrangement following the depletion of
DPB11 (Table 2). In each case, the breakpoint on chromosome
III corresponds with the only Ty retrotransposon on the left
arm and the rearrangement results in the loss of one copy of
the chromosome III centromere. We predict that this acentric
chromosome III fragment would be fused to the nonhomologous chromosome to allow this chromosome fragment to persist (Figure 5B). The chromosome fusion could result from an
interruption of a BIR event and subsequent resolution of the
strand invasion intermediate with a nonhomologous chromosome, resulting in a half crossover chromosome. Alternatively,
since the breakpoints on the nonhomologous chromosomes
coincide with Ty retrotransposon sites, it is possible that a homology-mediated repair mechanism, such as single-strand
annealing (SSA) (Mieczkowski et al. 2006), is involved in
the formation of this chromosome fusion (Figure 5B). This
mutant chromosome was confirmed by Southern blot analysis
of a representative illegitimate diploid isolated following the
depletion of NSE1 (Figure 6C, lanes 2 and 4). A probe specific
for chromosome III detected two chromosome sizes, one corresponding to intact chromosome III and another to the predicted size of the chromosome III–chromosome V fusion. We
also detected two chromosome bands following rehybridization with a probe specific for chromosome V, one corresponding to the size of intact chromosome V and the other
corresponding to the expected size of the chromosome fusion.
Finally, depletion of RFC2 resulted in one example of a “Hawthorne deletion” (class 3-6), an interstitial deletion between
repeated regions of the MATa and HMRa loci (Hawthorne
1963). We did not observe any examples of class 3-7, the
hallmark of which is amplification of sequences between FS1
and FS2 (Casper et al. 2009). The total ratio of the seven
rearrangement classes was 15:4:8:4:6:1:0 (3-1: 3-2: 3-3: 3-4:
3-5: 3-6: 3-7).
Boundaries of rearrangements correlate with Ty
retrotransposons, LTRs, tRNA genes, early replication
origins, and replication termination sites
To determine whether the boundaries of rearrangements are
correlated with particular genomic features, we performed
an enrichment test. We segmented the genome into 5-kb bins
and scored each bin for the presence or absence of genomic
features and breakpoints. For each feature, we determined the
fold enrichment of bins that contain both the feature and
a breakpoint in comparison to what would be expected if
breakpoints were randomly placed into bins (Table 3). Consistent with other studies of rearrangement breakpoints in
yeast (Dunham et al. 2002; Lemoine et al. 2005, 2008;
Argueso et al. 2008; Li et al. 2009), our boundaries of rearrangement were significantly enriched at loci with Ty retrotransposons, LTRs, and tRNA genes (Table 3). As these sites
Essential Genome Stability Genes
155
have repetitive sequences, they may represent endpoints of
resection that facilitate recombination repair between nonhomologous loci. We also found that boundaries of rearrangement were significantly enriched near early replication origins
and replication termination sites (Table 3). One possibility is
that misregulation of replication firing and replication fork
convergence causes double-stranded DNA breaks that promote rearrangement events.
Discussion
Comparison of conditional allele screens for genome
instability mutants
The collection of tetracycline-regulated promoter conditional alleles (Tet alleles) encompasses 773 essential genes
(63%), of a total of 1135 that are annotated in the SGD
(Mnaimneh et al. 2004; Yu et al. 2006). We quantified the
accumulation of Ddc2 damage foci in this set of strains after
first filtering for the 217 strains that showed accumulation
in S or G2 phase following promoter shutoff. We identified
47 genes that function to protect the genome from spontaneous DNA damage, 20 of which did not have previously
annotated roles in the maintenance of genome stability. A similar screen for Ddc2 foci accumulation was recently reported,
using a set of 592 temperature-sensitive conditional alleles
representing 399 essential genes (Li et al. 2011). Of the 114
genes that were in common to both sets of conditional alleles
(Figure S2A), mutants of 10 essential genes displayed elevated
levels of Ddc2 foci in both screening efforts, a small but significant overlap (P-value of 0.0043; hypergeometric test) (Figure
S2B, Table S4). Fifteen genes were identified in our screen that
were negative in Li et al. (2011) and 15 genes were identified
in Li et al. (2011) that were negative in our screen (Figure
S2B). Altogether, we identified 37 genes with elevated Ddc2
foci that were not identified by Li et al. (2011).
We also screened for the a-like faker chromosome instability phenotype in 208 Tet alleles that we assayed for Ddc2
foci formation. Of the 68 genes that were in common with
a recent a-like faker screening effort using ts alleles (Stirling
et al. 2011) (Figure S2C), the overlap of 11 essential gene
mutants with elevated levels of a-like fakers in both screening
efforts was insignificant (P-value of 0.064 by the hypergeometric test) (Figure S2D, Table S5). We identified 59 essential
genes that contribute to the suppression of genome instability
that were not identified by Stirling et al. (2011). Focusing on
the genes assayed in both screens, there were seven false
negatives in our screen of Tet alleles and 22 false negatives
in the Stirling et al. (2011) screen of 364 ts alleles. These, and
similar false negatives in the Ddc2 foci screens, likely represent cases where the false negative allele was not sufficiently
compromised to display a significant phenotype. Finally, a recent screen was performed to determine the extent of Rad52
foci in 305 essential chromosome instability genes. Comparison with our Ddc2 foci screen revealed that only the depletion
of CDC9, CDC45, MCM5, and NSE1 and PSF2 resulted in elevated levels of both Rad52 and Ddc2 foci (Stirling et al. 2012).
Given that each conditional allele collection is currently incomplete, that a positive score with one kind of allele is at
best weakly predictive of a positive score with a distinct allele,
that the overlap between screens of different allele collections
is small, and that the functions of essential genes are likely
perturbed to varying degrees within any one collection, screening complementary collections of different kinds of alleles will
ultimately be necessary to define the complete cohort of essential genes that maintain genome stability.
A common theme in the overlap among these screens of
essential gene collections is enrichment for genes that function
in DNA replication, indicating that among essential processes,
replication defects are strong contributors to genome instability. Both Ddc2 foci screens were enriched for genes with
DNA replication as their GO process annotations, relative to
the S. cerevisiae genome (14.5- and 25.3-fold enrichment for
this study and Li et al. 2011, respectively; Bonferroni corrected
P-values of 7.41 · 10212 and 2.51 · 10227). Similarly, both alike faker screens displayed enrichment for DNA replication
(10.8- and 17.5-fold enrichment for this study and Stirling
et al. 2011, respectively; Bonferroni corrected P-values of
1.87 · 10213 and 3.20 · 10219).
We compared the functional differences between essential gene alleles that had elevated Ddc2 foci and those that
had increased frequency of illegitimate mating, as this is the
first time the same set of essential genes has been analyzed
with both assays. Eighteen alleles displayed only elevated
levels of Ddc2 foci and 63 alleles had only the a-like faker
chromosome instability phenotype, while 29 alleles had elevated levels of both. This core of 29 genes was enriched for
DNA replication function relative to the genome (20.4-fold
enrichment; Bonferroni corrected P-value of 2.04 · 10212),
again indicating the primary role of replication errors in genome rearrangements. By contrast, the alleles that displayed
Table 3 Enrichment analysis of the correlation between boundaries of chromosome rearrangements (n = 14)
and selected genomic features
Feature
LTRs
tRNA genes
Ty retrotransposons
Termination sites
Early replication origins
High confidence ARSs
156
E. Cheng et al.
Fold enrichment
8.823
7.495
7.988
7.313
9.355
1.739
P-value
10212
1.87 ·
3.76 · 1029
0.005
0.007
0.0184
0.186
False discovery rate corrected P-value
3.55 · 10211
3.57 · 1028
0.0344
0.0333
0.0699
0.505
only the a-like faker chromosome instability were enriched for
genes involved in transcription initiation (15.9-fold enrichment; Bonferroni corrected P-value of 6.22 · 1026). By analyzing the overlap between the screens, we found that strains
with increased illegitimate mating tended to have a larger
fraction of cells with spontaneous Ddc2 foci, and that strains
with spontaneous Ddc2 foci formation above our cutoff were
more likely to have increased illegitimate mating, which suggests some predictive value of one phenotype for the other.
However, the overlap between the Ddc2 focus screen and the
a-like faker screen was far from absolute. Therefore, consistent with a recent study (Stirling et al. 2011), we suggest that
the complete set of genes with roles in genome maintenance
will be obtained not only by screening different allele collections, but also by the application of multiple screening
methods.
Essential genes involved in DNA replication are critical
for genome stability
We observed a prominent role for DNA replication genes in
the suppression of chromosome rearrangements. Defects in
a range of distinct replication functions, including initiation
(CDC45, DBF4, DPB11, MCM4, MCM5, MCM7, and PSF2),
elongation (CDC45, DNA2, MCM4, MCM5, MCM7, POL2,
POL30, PSF2, RFC2, and RFC5), and termination (UBC9)
caused spontaneous DNA damage and chromosome rearrangements, suggesting that each stage of replication is crucia to the maintenance of genome stability. The rearrangements
that we observed likely involve DNA double stranded breaks
(DSBs) and there are several mechanisms by which defects
in replication could contribute to DSB formation.
Reduced levels of proteins involved in prereplicative and
preinitiation complex formation at replication origins likely
result in fewer replication forks emanating from fewer
origins, increasing the likelihood that regions of the genome
might remain unreplicated and become susceptible to breakage. Consistent with this view, reduced levels of activated
origins of replication and elevated frequencies of gross
chromosomal rearrangements have been observed in strains
with mutations in CDC6, CLN2, ORC2, or SIC1 genes involved
in origin licensing (Bruschi et al. 1995; Lengronne and
Schwob 2002; Shimada et al. 2002; Tanaka and Diffley
2002; Bielinsky 2003). Depletion of DNA replication elongation factors might disrupt the kinetics of replication in S phase,
resulting in replication fork stalling and chromosome rearrangements as has been noted in RFA1 mutants (Chen
et al. 1998). Defects in elongation could also disrupt the coordinated movements of replisomes and transcription machinery along the DNA, leading to increased levels of collision and
DNA breakage (Deshpande and Newlon 1996; Ivessa et al.
2003) (Figure 7A). Another consequence of defective replication elongation could be the delay of Okazaki fragment synthesis resulting in the accumulation of single-stranded DNA
(ssDNA) on the lagging strand, secondary structure formation,
and blocks to replisome progression (Sogo et al. 2002) (Figure
7B). This type of mechanism has been proposed to allow the
Figure 7 Mechanisms by which genome instability occurs in replicationdeficient mutants. (A) tRNA genes and replication forks are clustered in
proximity to Ty retrotransposons. The transcription machinery creates
obstacles for replication fork progression and could lead to DSB formation
and resection to the repeated elements. (B) Secondary structure formation involving repeated Ty retrotransposons (arrows) on the lagging
strand causes replication fork stalling, subsequent replisome dissociation,
and the formation of double stranded breaks (DSBs). (C) Failure to resolve
termination structures at converging replication forks that flank Ty retrotransposons is a potential source of DSBs.
formation of hairpin structures at inverted Ty retrotransposon
repeats, causing chromosome rearrangements when DNA polymerase a and d are depleted (Lemoine et al. 2005, 2008;
Casper et al. 2009). Depletion of polymerase e (POL2) in our
study could cause fragile site instability by a similar mechanism. Finally, when two replication forks converge in the last
stages of replication, termination structures need to be accurately resolved prior to mitosis to prevent DSBs (Figure 7C).
Both Ubc9 and the Nse1-containing Smc5/6 complex have
connections to resolution of termination structures (Branzei
et al. 2006) and we observe that rearrangement breakpoints
are enriched at termination sites (Fachinetti et al. 2010) (Table
3), suggesting that defective termination could contribute to
accumulation of chromosome rearrangements. Together, our
results emphasize the importance of replication defects, in initiation, elongation, and termination, in causing DNA damage
and chromosome rearrangements.
Ty retrotransposons and tRNA genes promote
chromosome rearrangements
Each of the 38 rearrangement breakpoints that we mapped
in this study, regardless of the specific function of the gene
that was depleted, was proximal to a Ty retrotransposon
element, highlighting the critical role of these repetitive elements in chromosome rearrangements in yeast. Chromosome
rearrangements involving Ty retrotransposons are observed at
a basal level in wild-type S. cerevisiae strains and a number of
experimental connections between rearrangements and Ty
elements have been made (reviewed in Garfinkel 2005; Lesage
and Todeschini 2005; Mieczkowski et al. 2006).
Essential Genome Stability Genes
157
Ty retrotransposons could function in at least two ways to
promote chromosome rearrangements. They could represent sites of chromosome breakage (Lemoine et al. 2005)
or they could provide homologous sequences for recombination-mediated repair of breaks that occur at distal sequences (Hoang et al. 2010). The yeast fragile site FS2 is thought
to be a site of chromosome breakage, and a model in which
ssDNA at inverted pairs of Ty retrotransposons, such as FS2,
allows formation of secondary structures that inhibit the
progression of the replisome, causing replication fork stalling and DNA breakage has been proposed (Lemoine et al.
2005, 2008; Casper et al. 2009) (Figure 7B). However, of
the 38 chromosome rearrangement mutants that we mapped, we observed only 3 (7.9%) with boundaries within the
FS2 region, suggesting that other modes of chromosome
breakage predominate in our study.
Examination of the boundaries of chromosome rearrangements in replication mutants revealed significant enrichment
of nearby early replication origins and tRNA genes (Table 3).
Transcription complexes on tRNA genes can impede an oncoming replisome, thereby promoting replication fork pausing
and DSB formation (Figure 7A) (Deshpande and Newlon
1996; Ivessa et al. 2003), suggesting that additional breakage
could result from transcription–replication collisions. This
combination of features surrounding breakpoints of rearrangement is consistent with those observed at natural evolutionary
breakpoints when S. cerevisiae is compared to related yeasts,
as well as breakpoints observed during artificial evolution of S.
cerevisiae (Dunham et al. 2002; Kellis et al. 2003; Di Rienzi
et al. 2009), suggesting that in addition to replication fidelity,
these features are important determinants of instability.
Parallels with human common fragile sites
There are a number of parallels between common fragile
sites in yeast and in humans. Inhibition or depletion of DNA
polymerases (Glover et al. 1984; Lemoine et al. 2005, 2008)
or DNA damage checkpoint proteins (Casper et al. 2002;
Cha and Kleckner 2002; Arlt et al. 2004; Schwartz et al.
2005; Durkin et al. 2006; Raveendranathan et al. 2006;
Vernon et al. 2008; Focarelli et al. 2009) can induce chromosome breaks at common fragile sites in both yeast and
human. Although human common fragile sites lack distinctive sequence similarities, they have attributes that impair
replication progression (Glover et al. 1984, 2005; Zlotorynski
et al. 2003), a shared property of yeast fragile sites (Roeder and
Fink 1980; Deshpande and Newlon 1996; Cha and Kleckner
2002; Ivessa et al. 2003; Lemoine et al. 2005; Admire et al.
2006; Raveendranathan et al. 2006). Additionally, recent studies of the human common fragile site FRA3B have suggested
that instability at this site is not due to replication fork slowing
or stalling, but rather is due to a paucity of replication initiation
events (Letessier et al. 2011). In our studies, early firing origins
of replication are enriched in regions with rearrangement
breakpoints. Depletion of replication initiation factors could
disrupt origin firing at these sites and thereby contribute to
instability in a manner analogous to FRA3B. It will be of
158
E. Cheng et al.
great interest to test the general role of replication proteins
in suppressing chromosome rearrangements that we have
observed in yeast in the maintenance of human common
fragile sites.
Acknowledgments
We thank Philip Hieter for strains, Tao Qi for technical
assistance, Lisa Yu for strain construction, and all members
of the Brown laboratory for insightful discussions. This research was supported by the Canadian Institutes of Health
Research (grant MOP-79368 to G.W.B.), the Canada Excellence Research Chair Program (F.P.R.), the Canadian Institute
for Advance Research Fellowship (F.P.R.), and the Natural
Sciences and Engineering Research Council of Canada (J.A.V.).
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Communicating editor: J. Nickoloff
GENETICS
Supporting Information
http://www.genetics.org/content/suppl/2012/06/05/genetics.112.141051.DC1
Genome Rearrangements Caused by Depletion
of Essential DNA Replication Proteins
in Saccharomyces cerevisiae
Edith Cheng, Jessica A. Vaisica, Jiongwen Ou, Anastasia Baryshnikova, Yong Lu,
Frederick P. Roth, and Grant W. Brown
Copyright © 2012 by the Genetics Society of America
DOI: 10.1534/genetics.112.141051
FIGURE S1
A
SRD1
PET18
delta
CWH43
Ty retrotransposon
Ty retrotransposon
MAK32
MAK31
Fragile site 2: FS2-2
FEN2
ARS310
YCRWdelta11 YCR028C-A
tQ(UUG)C
RHB1
YCR026C
Ty retrotransposon
Ty retrotransposon
FEN2
X
E XEE
E
E
XE
Southern blot fragments
FS2-2
FEN2
B
10000
8000
6000
5000
4000
3500
3000
2500
2000
1500
FIGURE S1. FS2
Eco E
Xba X
FEN2
Ty1
2 SI EcoRI + XbaI
XbaI
EcoRI
EcoRI + XbaI
XbaI
EcoRI
Fragile site 1:
FS2
E. Cheng et al. S. cerevisiae
FS2-2
E. Cheng et al. 3 SI 4 SI E. Cheng et al. Tables S1-­‐S8 Supporting Tables Tables S1-­‐S8 are available for download at http://www.genetics.org/content/suppl/2012/06/05/genetics.112.141051.DC1. Table S1. Quantification of Ddc2 foci in Tet allele strains Table S2. GO function and GO process annotations of genes whose depletion results in elevated levels of Ddc2 foci Table S3. a-­‐like faker scores from patch mating test with Tet allele strains Table S4. Comparison of Tet alleles and ts-­‐alleles with elevated levels of Ddc2 foci Table S5. Comparison of Tet alleles and ts-­‐alleles with a-­‐like faker phenotype Table S6. Strain Genotypes Table S7. Primers used to generate probes in Southern blot analysis Table S8. Primers used for PCR amplification and sequencing of FS1 and FS2 E. Cheng et al. 5 SI